Hi, I am making fusion gene with overlapping splice extension PCR method. A,B are primer pairs of HA gene, C,D are ones of Fc gene, and B shares complementary 50 bases to C(this 50-bases encoding linker).
My story is weird as below:
1. I can amplify HA and Fc, respectively.
2. I can also amplify HA-linker-Fc with undiluted HA and Fc PCR products from 1 as template, and A, D as primer pairs.
3.(!!!!!) I can not re-amplify HA-linker-Fc fusion gene with gel purified HA-linker-Fc from above 2!!!!
I try to optimize PCR conditions, such as serially dilulting HA-LINKER-Fc in 1:10, 1:100, 1:1000 as templates. However, whatever I do, I can not get the fusion gene reamplified at all!!!!
I am dying, who can help me?
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